The detection of specific nucleic acids is an important tool for diagnostic medicine and molecular biology research. Gene probe assays currently play a role in a number of spheres of diagnostic medicine and molecular biology, including for example identifying infectious organisms such as bacteria and viruses, in probing the expression of normal and mutant genes and identifying genes associated with disease or injury, such as oncogenes, in typing tissue for compatibility preceding tissue transplantation, in matching tissue or blood samples for forensic medicine, for responding to emergency response situations like a nuclear incident or pandemic flu outbreak, in determining disease prognosis or causation, and in exploring homology among genes from different species.
Ideally, a gene probe assay should be sensitive, specific and easily automatable. The requirement for sensitivity (i.e. low detection limits) has been greatly alleviated by the development of the polymerase chain reaction (PCR) and other amplification technologies which allow researchers to exponentially amplify a specific nucleic acid sequence before analysis. For example, multiplex PCR amplification of SNP loci with subsequent hybridization to oligonucleotide arrays can be used to simultaneously genotype hundreds of SNPs.
Specificity is a challenge for gene probe assays. The extent of molecular complementarity between probe and target defines the specificity of the interaction. Variations in composition and concentrations of probes, targets and salts in the hybridization reaction as well as the reaction temperature, and length of the probe may all alter the specificity of the probe/target interaction. It can be possible under some circumstances to distinguish targets with perfect complementarity from targets with mismatches, although this is generally difficult using traditional technology, since small variations in the reaction conditions will alter the hybridization. Techniques for mismatch detection include probe digestion assays in which mismatches create sites for probe cleavage, and DNA ligation assays where single point mismatches prevent ligation.
A variety of enzymatic and non-enzymatic methods are available for detecting sequence variations. Examples of enzyme based methods include Invader™, oligonucleotide ligation assay (OLA) single base extension methods, allelic PCR, and competitive probe analysis (e.g. competitive sequencing by hybridization). Enzymatic DNA ligation reactions are well known in the art and have been used extensively in SNP detection, enzymatic amplification reactions and DNA repair. A number of non-enzymatic or template mediated chemical ligation methods can also be used to detect sequence variations. These include chemical ligation methods that utilize coupling reagents, such as N-cyanoimidazole, cyanogen bromide, and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride.
A widely recognized problem when analyzing RNA for genetic studies is the inherent instability of the RNA itself. RNA naturally has a short lifetime in living organisms because organisms regulate the RNA concentration which regulates downstream processes which are dependent on the RNA. There are also many natural processes which lead to the destruction of an RNA.
In recent years researchers have spent considerable effort developing methods for the analysis of gene expression in cells. This is generally accomplished by analyzing the cell contents for the amount of specific mRNA molecules present. Measurements of gene expression are based on the underlying assumption that the analyzed RNA sample closely resembles the number of transcripts in vivo. Hence, maintaining the integrity of the RNA after extraction from the cell is of paramount importance. Researchers have recognized that transcripts of different genes (mRNA) possess different stabilities which implies that that degradation of RNA occurring during the isolation procedure may be non-uniformly distributed among different RNA molecules. One comparison of RNA samples of different degrees of degradation shows that up to 75% of microarray-based measurements of differential gene expression can be caused by degradation bias. Auer H, Liyanarachchi S, Newsom D, Klisovic M I, Marcucci G, Kornacker K (2003), Nature Genetics 35:292-293.
There remains a need for methods and compositions for efficient and specific nucleic acid detection and for stabilization of RNA following extraction from cells.